The activities of steroid receptors (SR) are regulated both by hormones and by alterations in cell signaling pathways. Our goal is to understand how phosphorylation regulates the activity of the human progesterone receptor (PR) :Specific Aim #l: To utilize state of the art, high sensitivity mass spectrometry to identify additional phosphorylation sites and/or other post- translational modifications in PR. Specific Aim #2. To develop and characterize novel phosphorylation site-specific antibodies for analysis of the role of phosphorylation in receptor structure and function. We have already produced two phosphorylation site-specific antibodies and applied them to an analysis of cross-talk with PR. These antibodies and the new ones to be made in this aim will permit uniquely powerful approaches to studying regulation of PR phosphorylation. Specific Aim #3: To assess the role of phosphorylation in specific receptor functions. We will test our hypothesis that two important functions of PR phosphorylation are l: to regulate transcriptional activity through modulation of protein/protein interactions (both intramolecular and intermolecular) and 2: to regulate receptor turnover. Specific Aim #4: To examine the hypothesis that an additional important function for phosphorylation is to integrate PR with other signaling pathways, we will use novel phosphorylation site specific antibodies as probes for specific phosphorylations, we will elucidate the cell signaling pathways that regulate PR phosphorylation and activity. Modulation of cell signaling pathways alters PR activity and can even cause antagonists to act as agonists, but the pathways and mechanisms by which this occurs are not well understood. We will use the phosphorylation site specific antibodies as an aid in defining the pathways and kinases responsible for specific phosphorylations, leading to altered receptor activation and to examine cell cycle effects on receptor phosphorylation and function. Understanding the interplay between steroid receptors and cell signaling pathways will aid in understanding the normal actions of agonists and antagonists as well as the changes that occur in diseases such as hormone dependent cancer where cell signaling is altered.